GPR68 is an understudied G-protein coupled receptor (GPCR), responds to protons and couples to multiple signaling pathways. It is expressed in many cells and tissues and believed to play important but presently unclear roles in normal physiology and pathological conditions. While GPR68 is expressed most abundantly in the brain, its potential function in this organ are yet to be identified. Pharmacological studies with GPR68 have been difficult and controversial, mainly due to the lack of small molecule tool compounds. We took a combined approach (modeling and virtual screening, SAR, and cell-based functional assays) and successfully identified the first Gs-based positive allosteric modulator (PAM), ogerin, for GPR68. We were the first to show that ogerin suppresses learning and memory in the wild-type but not GPR68 KO mice, suggesting an important role in the brain function (Huang et al., 2015). Based on our preliminary results, we are proposing to take the same combined approach, but at much larger scale, to design and develop functionally selective (biased) allosteric modulators for individual signaling pathways mediated by protons at GPR68. Specifically, we propose to identify and optimize positive and/or negative allosteric modulators (PAMs and/or NAMs) with distinct allosteric activity (?, -values) for the following pathways (readout): Gs-cAMP (with cAMP production and CRE-Luc reporter), Gq-calcium release (Calcium release on FLIPR), Gq-PI hydrolysis (SAP PI hydrolysis), G12/13 Rho (G12/13 specific SRE-Luc reporter), hypoxia signaling pathway (HRE-Luc reporter). The proposal has three components: (i) modeling and virtual screening will be carried out in Dr. Brian Shoichet lab (UCSF); (ii) chemistry synthesis and structural modification will be carried out in Dr. Jian Jin lab (Mount Sinai School of Medicine), and (iii) cell-based functional assays and compound characterization will be carried out in Dr. Xi-Ping Huang lab and Dr. Terry Kenakin lab (UNC Chapel Hill). Dr. Xi-Ping Huang is a Research Assistant Professor in Dr. Bryan Roth lab and serves as the Assistant Scientific Director for NIMH PDSP (National Institute of Mental Health Psychoactive Drug Screening Program). The SAR studies will be guided by novel and quantitative analysis for induced functional selectivity (10?Log(?)) between two selected signaling pathways (assays) and assisted by modeling/docking studies. 1 ? 2 candidate modulators of each type (?-, -, ?-PAMs and/or -NAMs) for each individual signaling pathways will be selected for (i) selectivity assays over related proton receptors GPR4 and GPR65; (ii) binding activity at >50 common drug targets; (iii) off-target agonist activity at 318 human GPCRs (90% of human GPCRome); (iv) hERG inhibition and Caco-2 assays. At the end of the proposed project, we hopefully will identify potent, pathway specific, and functionally selective allosteric modulators as pharmacological probes for GPR68 studies.